Erythropoietin, but not the correction of anemia alone, protects from chronic kidney allograft injury.

Anemia can contribute to chronic allograft injury by limiting oxygen delivery to tissues, particularly in the tubulointerstitium. To determine mechanisms by which erythropoietin (EPO) prevents chronic allograft injury we utilized a rat model of full MHC-mismatched kidney transplantation (Wistar Furth donor and Lewis recipients) with removal of the native kidneys. EPO treatment entirely corrected post-transplant anemia. Control rats developed progressive proteinuria and graft dysfunction, tubulointerstitial damage, inflammatory cell infiltration, and glomerulosclerosis, all prevented by EPO. Normalization of post-transplant hemoglobin levels by blood transfusions, however, had no impact on chronic allograft injury, indicating that EPO-mediated graft protection went beyond the correction of anemia. Compared to syngeneic grafts, control allografts had loss of peritubular capillaries, higher tubular apoptosis, tubular and glomerular oxidative injury, and reduced expression of podocyte nephrin; all prevented by EPO treatment. The effects of EPO were associated with preservation of intragraft expression of angiogenic factors, upregulation of the anti-apoptotic factor p-Akt in tubuli, and increased expression of Bcl-2. Inhibition of p-Akt by Wortmannin partially antagonized the effect of EPO on allograft injury and tubular apoptosis, and prevented EPO-induced Bcl-2 upregulation. Thus non-erythropoietic derivatives of EPO may be useful to prevent chronic renal allograft injury.

Prolonged cold ischemia accelerates cellular and humoral chronic rejection in a rat model of kidney allotransplantation.

One of the leading causes of long-term kidney graft loss is chronic allograft injury (CAI), a pathological process triggered by alloantigen-dependent and alloantigen-independent factors. Alloantigen-independent factors, such as cold ischemia (CI) may amplify the recipient immune response against the graft. We investigated the impact of prolonged cold ischemia and the subsequent delayed graft function on CAI in a fully MHC-mismatched rat model of kidney allotransplantation. Prolonged CI was associated with anticipation of proteinuria onset and graft function deterioration (ischemia: 90d; no ischemia: 150d), more severe tubular atrophy, interstitial fibrosis, and glomerulosclerosis, and increased mortality rate (180d survival, ischemia: 0%; no ischemia: 67%). In ischemic allografts, T and B cells were detected very early and were organized in inflammatory clusters. Higher expression of BAFF-R and TACI within the ischemic allografts indicates that B cells are mature and activated. As a consequence of B cell activity, anti-donor antibodies, glomerular C4d and IgG deposition, important features of chronic humoral rejection, appeared earlier in ischemic than in non-ischemic allograft recipients. Thus, prolonged CI time plays a main role in CAI development by triggering acceleration of cellular and humoral reactions of chronic rejection. Limiting CI time should be considered as a main target in kidney transplantation.

Rabbit anti-rat thymocyte immunoglobulin preserves renal function during ischemia/reperfusion injury in rat kidney transplantation.

Ischemia/reperfusion (I/R) injury is an important cause of renal graft dysfunction in humans. Increases in cold and warm ischemia times lead to a higher risk of early post-transplant complications including delayed graft function and acute rejection. Moreover, prolonged cold ischemia is a predictor of long-term kidney graft loss. The protective effect of rabbit anti-rat thymocyte immunoglobulin (rATG) was evaluated in a rat model of I/R injury following syngeneic kidney transplantation. Serum creatinine concentration was evaluated at 16 h and 24 h post-transplant. Animals were sacrificed 24 h post-transplant for evaluation of histology, infiltrating leukocytes, nitrotyrosine staining, and apoptosis. rATG was effective in preventing renal function impairment, tissue damage and tubular apoptosis associated with I/R only when was given 2 h before transplantation but not at the time of reperfusion. Pretransplant rATG treatment of recipient animals effectively reduced the amount of macrophages, CD4(+), CD8(+) T cells and LFA-1(+) cells infiltrating renal graft subjected to cold ischemia as well as granzyme-B expression within ischemic kidney. On the other hand, granulocyte infiltration and oxidative stress were not modified by rATG. If these results will be translated into the clinical setting, pretransplant administration of Thymoglobuline(®) could offer the additional advantage over peri-transplant administration of limiting I/R-mediated kidney graft damage.

Brain lipid composition in grey-lethal mutant mouse characterized by severe malignant osteopetrosis.

The grey-lethal mouse (gl/gl) mutant most closely resembles the severe human malignant autosomal recessive OSTM1-dependent form of osteopetrosis that it has been described to be associated with neurological abnormalities. For this reason, we have analyzed the brain lipid composition (gangliosides, neutral glycosphingolipids, phospholipids and cholesterol), from gl/gl mice at different ages of development and compared with wild type mice. Both cholesterol and glycerophospholipid content and pattern in the gl/gl and control mice were very similar. In contrast, significant differences were observed in the content of several sphingolipids. Higher amount of the monosialogangliosides GM2 and GM3, and lower content of sphingomyelin, sulfatide and galactosylceramide were observed in the gl/gl brain with respect to controls. The low content of sphingomyelin, sulfatide and galactosylceramide is consistent with the immunohistochemical results showing that in the grey-lethal brain significant depletion and disorganization of the myelinated fibres is present, thus supporting the hypothesis that loss of function of the OSTM1 causes neuronal impairment and myelin deficit.

Extracellular vesicles derived from T regulatory cells suppress T cell proliferation and prolong allograft survival.

We have previously shown that rat allogeneic DC, made immature by adenoviral gene transfer of the dominant negative form of IKK2, gave rise in-vitro to a unique population of CD4+CD25- regulatory T cells (dnIKK2-Treg). These cells inhibited Tcell response in-vitro, without needing cell-to-cell contact, and induced kidney allograft survival prolongation in-vivo. Deep insight into the mechanisms behind dnIKK2-Treg-induced suppression of Tcell proliferation remained elusive. Here we document that dnIKK2-Treg release extracellular vesicles (EV) riched in exosomes, fully accounting for the cell-contact independent immunosuppressive activity of parent cells. DnIKK2-Treg-EV contain a unique molecular cargo of specific miRNAs and iNOS, which, once delivered into target cells, blocked cell cycle progression and induced apoptosis. DnIKK2-Treg-EV-exposed T cells were in turn converted into regulatory cells. Notably, when administered in-vivo, dnIKK2-Treg-EV prolonged kidney allograft survival. DnIKK2-Treg-derived EV could be a tool for manipulating the immune system and for discovering novel potential immunosuppressive molecules in the context of allotransplantation.

Induction of axonal differentiation by silencing plasma membrane-associated sialidase Neu3 in neuroblastoma cells.

A reduction of 70% of the plasma membrane-associated sialidase Neu3 activity, due to a corresponding reduction of the enzyme expression by transducing cells with a short hairpin RNA encoding a sequence target (complementary messenger of mouse Neu3), caused neurite elongation in Neuro2a murine neuroblastoma cells. The differentiation process was accompanied in parallel by an increase of the acetylcholinesterase activity, a moderate increase of the c-Src expression and by the presence of the axonal marker tau protein on the neurites. The sphingolipid pattern and turnover in transduced and control cells were characterized by thin layer chromatography, mass spectrometry and metabolic radiolabeling after feeding cells with tritiated sphingosine. Control cells contained about 2 nmol of gangliosides/mg cell protein. GM2 was the main compound, followed by GD1a, GM3 and GM1. In Neu3 silenced cells, the total ganglioside content remained quite similar, but GM2 increased by 54%, GM3 remain constant, and GM1 and GD1a decreased by 66% and 50%, respectively. Within the organic phase sphingolipids, ceramide decreased by 50%, whereas the sphingomyelin content did not change in Neu3 silenced cells.